title: Construction of a microfluidic chip, using dried-down reagents, for LATE-PCR amplification and detection of single-stranded DNA.
abstract: LATE-PCR is an advanced form of non-symmetric PCR that efficiently generates single-stranded DNA which can readily be characterized at the end of amplification by hybridization to low-temperature fluorescent probes. We demonstrate here for the first time that monoplex and duplex LATE-PCR amplification and probe target hybridization can be carried out in double layered PDMS microfluidics chips containing dried reagents. Addition of a set of reagents during dry down overcomes the common problem of single-stranded oligonucleotide binding to PDMS. These proof-of-principle results open the way to construction of inexpensive point-of-care devices that take full advantage of the analytical power of assays built using LATE-PCR and low-temperature probes.
authors.affiliation: Department of Biology, Brandeis University, 415 South Street, Waltham, MA 02454, USA. wangh@brandeis.edu.
authors.forename: Yanwei
authors.initials: Y
authors.lastname: Jia
authors.forename: Pui-In
authors.initials: PI
authors.lastname: Mak
authors.forename: Conner
authors.initials: C
authors.lastname: Massey
authors.forename: Rui P
authors.initials: RP
authors.lastname: Martins
authors.forename: Lawrence J
authors.initials: LJ
authors.lastname: Wangh
mesh_terms: D004277:DNA, Single-Stranded / Q000032:analysis*; D004129:Dimethylpolysiloxanes / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation*; D009693:Nucleic Acid Hybridization; D019095:Point-of-Care Systems; D016133:Polymerase Chain Reaction / Q000295:instrumentation*; D013696:Temperature
chemical_list: D004277:DNA, Single-Stranded; D004129:Dimethylpolysiloxanes; D005456:Fluorescent Dyes; C013830:baysilon